In stating a vision for the future in “Biobased Industrial Products, Priorities for Research and Commercialization”, the National Research Council has proposed U.S. leadership for the global transition to biobased products. The report provides compelling evidence for a competitively priced biobased products industry that will eventually replace much of the petrochemical industry. In light of this report and the desirability of reducing U.S. reliance on foreign oil, there is an increasing interest in generating commodity and fine chemicals from widely available U.S. renewable resources, e.g., crops, through fermentation. In the last few years, companies have invested hundreds of millions of dollars in commercializing the microbial production of several biochemicals, such as lactic acid.
Microbial fermentation processes are used to generate a wide variety of important biochemicals such as ethanol and lysine (markets in the billions of U.S. dollars). In order to be economic, fermentations rely on microorganisms which have been developed by selection or genetic means to accumulate a specific product that is produced via metabolism. Microbial metabolic pathways are not naturally optimal for the generation of a desired chemical, but have instead evolved for the benefit of the organism. Metabolic engineering is the targeted and rational alteration of metabolism, and it involves the redirection of cellular activities to generate a new product or generate a product at a higher rate or yield.
Pyruvate (pyruvic acid) is a three-carbon ketoacid synthesized at the end of glycolysis. Pyruvate is an important raw material for the production of L-tryptophan, L-tyrosine, 3,4-dihydroxyphenyl-L-alanine, and for the synthesis of many drugs and biochemicals. Pyruvate has use in the chemical industry and finds wide application in cosmetics. Clinical studies have found that pyruvate can promote weight loss and fat loss, hence it is commonly marketed in tablet form as a dietary supplement. Recent research indicates that pyruvate also functions as an antioxidant, inhibiting the production of harmful free radicals.
Certain microorganisms have been found to produce useful quantities of pyruvate from glucose, an inexpensive substrate derived from corn starch. The yeasts Debaryomyces coudertii (M. Moriguchi, Agr. Biol. Chem. 46: 955-961 (1982)) and Saccharomyces exiguus (A. Yokota et al., Agr. Biol. Chem. 48: 2663-2668 (1984)), for example, are known to accumulate pyruvate, as are the basidiomycetes Schizophyllum commune (S. Takao et al., J. Ferm. Tech. 60: 277-280 (1982)), and Agricus campestris (A. Yokota et al., Agr. Biol. Chem. 48: 2663-2668 (1984)). The yeast strain Torulopsis glabrata IFO 0005 was found to be a superior strain for the production of pyruvate (T. Yonehara et al., J Ferm. Bioeng. 78: 155-159 (1994)), accumulating 67.8 g/L pyruvate in 63 hours (yield 0.494) in a fed-batch fermentation with successive additions of glucose (R. Miyata et al., J. Ferm. Bioeng. 82: 475-479 (1996)). A higher yield (0.673) of pyruvate was observed in T. glabrata ACII-33, a mutant with decreased pyruvate decarboxylase (PDC) activity (R. Miyata et al., J. Biosci. Bioeng. 88: 173-177 (1999)). Decreased PDC activity prevented the formation of acetate via acetaldehyde and thus increased the pyruvate production. T. glabrata ACII-33 accumulated 60.3 g/L pyruvate in 47 hours in a 3 L jar fermenter.
Bacteria of the genera Corynebacterium (A. Yokota et al., Agr. Biol. Chem. 48: 2663-2668 (1984)) and Acinetobacter (Y. Izumi et al., Agr. Biol. Chem. 46: 2673-2679 (1982)), Enterobacter aerogenes (A. Yokota et al., Agr. Biol. Chem. 48: 705-711 (1989)), and Escherichia coli (A. Yokota et al., Appl. Microbiol. Biotech. 41: 638-643 (1994)) are also known to accumulate pyruvate. A lipoic acid auxotroph of E. coli (strain W14851ip2) was found to produce pyruvate aerobically from glucose under lipoic acid deficient conditions. This strain accumulated 25.5 g/L pyruvate in 32 hours with a yield of 0.51 in polypepton (4 g/L) supplemented media (A. Yokota et al., Appl. Microbiol. Biotech. 41: 638-643 (1994)).
Alanine, which is derived from pyruvate, is an alpha amino acid that is also commercially important, for example as a starting material in the chemical industry. L-alanine is a chiral building block being one of the smallest chiral compounds, with four important functional groups: hydrogen, methyl, amino, and carboxylic acid. Presently, alanine is produced using metabolically engineered Corynebacterium or Brevibacterium bacterial strains in which alanine dehydrogenase is overexpressed. However, the efficiency of this method of production is limited because large quantities of carbon move from pyruvate to acetyl-CoA and therefore are unavailable for alanine production.
Diacetyl, also derived from pyruvate, is a flavoring/texture agent for dairy products, and could find additional use in food products.
These compounds (pyruvate, alanine and diacetyl) have current market prices from $10 to $50/pound. Improved production methods from renewable resources would open new markets for pyruvate and its derivatives and thus reduce reliance on petroleum-derived products.